Mycoplasmosis is usually subtle, but Mycoplasma alligatoris causes acute lethal primary infection of susceptible hosts. The goal of this work is to explain the mechanisms and evolutionary origins of that virulence. A genome survey indicated that M. alligatoris uses sialidase (Nanl) and hyaluronidase (NagH) to generate fuel for glycolysis from host cell glycans. Its attenuated sibling species Mycoplasma crocodyli possesses NagH but not Nanl, so damage to the host's extracellular matrix alone cannot explain the particular virulence of M. alligatoris. While nan and nag systems could contribute to advantages in nutrient acquisition, our hypothesis is that virulence is a consequence of synergy of those glycosidases, co- localized by infection, that potentiates host CD44-transduced apoptosis, necrosis, and inflammation consistent with the fulminant disease observed. The corollary hypothesis is that a fitness-enhancing operon like nan can precipitate virulence after horizontal transfer to a genome like M. crocodyli's which is pre- adapted with nag, a significant paradigm for the emergence of pathogens. This proposal focuses on the nan and nag glycosidases and their interactions in naturally-simplified small-genome pathogens, and hosts which represent a significant stage in the history of co-evolution of microbial pathogens and vertebrate immunity. Our aims are to: 1) Demonstrate the advantages of the nan and nag systems to M. alligatoris for fitness reflected in growth rate and nutrient flux, by transposon-mediated knockout and complementation of genes for the key intermediary enzymes unique to their respective pathways;2) Define the cytopathic effects of the glycosidases reflected in Fas (CD95), NO, and IFN expression, by inactivation and complementation of nanl and nagH using in vitro primary fibroblast infection models;and 3) Annotate the M. crocodyli genome and model the effects of horizontal transfer of nan genes on fitness and virulence of M. crocodyli as described for M. alligatoris. The results are expected to provide direct evidence of linkage between fitness and virulence factors of M. alligatoris, and support horizontal transfer of systems favorable by selection for nutritional fitness as an evolutionary mechanism precipitating the virulence of some bacteria. Relevance: Beyond explaining the remarkable pathogenicity of M. alligatoris, this work will investigate how CD44 signaling is modulated by bacteria during infection, which could suggest new treatment modalities for many invasive pathogens. Assessing the importance of genome pre-adaptation with modestly advantageous genes like hyaluronidase to precipitation of severe virulence following horizontal transfer of a system like nan could lead to predictive models for the emergence and evolution of pathogens.